Thrombin is the product of the activation of prothrombin by Factor Xa in plasma. It is a potent broadly specific serine proteinase that converts fibrinogen to fibrin and promotes fibrin cross-linking by activating Factor XIII. Amongst a number of other observed biological activities, thrombin also controls several feedback loops in the clotting cascade and induces the platelet release reaction (1, 2).
Thrombin has been used as a topical haemostatic agent for many years. However, it is as a component of fibrin sealant (fibrin glue) that the clinical use of thrombin is likely to expand. Thrombin is used in fibrin sealant to convert fibrinogen to fibrin on a cut surface or within a graft and numerous surgical applications have been described in a wide range of surgical specialities (3, 4).
Bovine thrombin is currently used widely as a topical haemostatic agent or as a component of commercial fibrin sealant products. While such thrombin products are biologically effective, they are associated with well-documented risk of allergic responses and induction of antibodies to the bovine thrombin or to impurities such as bovine factor V, usually after repeat use (5, 6 and 7). Ortel et al (8) recently concluded that such acquired coagulation factor inhibitors probably occur more commonly than is currently appreciated and although frequently clinically benign, these inhibitors may be associated with life-threatening haemorrhage. For this reason the development of a process to produce human thrombin suitable for use as a topical haemostat or for inclusion in a fibrin sealant product, has been sought.
Intrinsically, thrombin is formed when prothrombin (Factor II) is converted by activated Factor X, activated a Factor V, phospholipid and calcium ions into thrombin. Conversion of prothrombin to thrombin can occur without some of the associated components, however, the rate of conversion is undesirably slow.
There are three main in vitro prothrombin conversion methods known in the art. The first method relies on the use of thromboplastin. Prothrombin is converted to thrombin using thromboplastin preferably in the presence of calcium chloride. This is described in a number of patent specifications such as EP 0439156A and EP 0505604A. A disadvantage of this method is that the thromboplastin is usually a crude preparation which has been prepared from freshly homogenised brain, lung or intestinal tissue. This procedure is not appropriate for the preparation of human thrombin as the reagents, depending on their source, can carry the risk of virus or cross-species contamination.
A second method utilises some components of snake venom to yield thrombin (9, 10, 11). However, it has been reported that some of the venoms do not cleave the same bonds within prothrombin, as the natural activator, Factor Xa (12). Thus, there may be dangerous implications should a non-physiological form of thrombin be used clinically.
The third in vitro method is essentially the same as the intrinsic in vivo process, wherein prothrombin is converted to thrombin by activated Factor X, Factor V, phospholipid and calcium ions under near physiological conditions. This has been described, for instance in, EP 0528701, EP 0378798 and U.S. Pat. No. 5,219,995. However, the thrombin produced is often unstable unless exogenous proteins, polyols and/or sugars are added to the thrombin to stabilise it.
Since human thrombin is derived from plasma obtained from blood donations, there is a risk of contamination of the thrombin by any viruses present in the original blood donation. Thus, any human thrombin preparation designed for clinical use, should be subjected to a virus inactivation step, prior to use.
Virus-inactivation by solvent-detergent treatment has been described previously (13). However, the thrombin preparation may need to be subjected to further purification steps in order to remove the solvent-detergent. Other workers have described the use of virus/inactivated prothrombin feedstocks, but have not described methods for virus/inactivation of the thrombin products prepared from them, for example EP 0378798 and EP 0543178. Terminal (e.g. a final step of a process) viral-inactivation of the product is viewed as probably the safest and most effective method of virus/inactivation, as it minimises the chance of recontamination.
There is thus a requirement in the art to produce thrombin which is terminally virus/inactivated, especially by heat-treatment.
Generally speaking the present invention is based on the surprising discovery that prothrombin can be converted to thrombin in good yield, under acidic conditions and that these acidic conditions promote the stability of the thrombin generated.